昆虫保幼激素受体的研究与应用进展

保幼激素(juvenile hormone, JH)是昆虫内分泌系统中的关键激素之一,对昆虫生长发育、变态、繁殖起着重要的调控作用。近年来有关JH的分子作用机制取得了极大的进展,主要得益于JH受体的鉴定,大量研究表明JH可通过胞内受体和膜受体两个途径来发挥生理调控功能。本文将从JH胞内受体Met的发现及鉴定、Met转录活性的调控因素、Met功能研究进展,以及Met作为JH受体在JH激动剂及拮抗剂筛选中的应用等方面对JH胞内受体的研究进展进行重点阐述;同时综述了有关JH膜受体的信号通路以及膜受体与核受体的互作等方面的研究进展。     

Juvenile hormone modulates 20-hydroxyecdysone-inducible ecdysone receptor and ultraspiracle gene expression in the tobacco hornworm, Manduca sexta

 Insect molting and metamorphosis are orchestrated by ecdysteroids with juvenile hormone (JH) preventing the actions of ecdysteroids necessary for metamorphosis. During the molt and metamorphosis of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, the isoforms involved in the ecdysone receptor (EcR)/Ultraspiracle (USP) complex change with the most dramatic switch being the loss of USP-1 and the appearance of USP-2 during the larval and pupal molts. We show here that this switch in USP isoforms is mediated by high 20-hydroxyecdysone (20E) and that the presence of JH is necessary for the down-regulation of USP-1 mRNA. The decrease of USP-1 mRNA in day 2 fourth instar larval epidermis in vitro required exposure to a high concentration (10^–5 M) of 20E equivalent to the peak ecdysteroid concentration in vivo, whereas the increase of USP-2 mRNA occurred at lower concentrations (effective concentrations, EC₅₀=6.3×10^–7 M). During the pupal molt of allatectomized larvae which lack JH, USP-2 mRNA increased normally with the increasing ecdysteroid titer, whereas USP-1 mRNA remained high until pupation. When day 2 fifth instar larval epidermis was exposed to 500 ng/ml 20E in the absence of JH to cause pupal commitment of the cells by 24 h, USP-1 RNA remained at its high preculture level for 12 h, then increased two- to threefold by 24 h. The increase was prevented by the presence of 1 μg/ml JH I which also prevents the pupal commitment of the cells. By contrast, USP-2 mRNA increased steadily with the same EC₅₀ as in fourth stage epidermis, irrespective of the presence or absence of JH. Under the same conditions, mRNAs for both EcR-B1 and EcR-A isoforms were up-regulated by 20E, each in its own time-dependent manner, similar to that seen in vivo. These initial mRNA increases were unaffected by the presence of JH I, but those seen after 12 h exposure to 20E were prevented by JH, indicating a difference in response between larvally and pupally committed cells. The presence of JH which maintained larval commitment of the cells also prolonged the half-life of the EcR proteins in these cells. These results indicate that both EcR and USP RNAs are regulated by 20E and can be modulated by JH in a complex manner with only that of USP-2 apparently unaffected. Received: 16 July 1998 / Accepted: 5 August 1998     

Estradiol inhibits the effects of extracellular ATP in human sperm by a non genomic mechanism of action

Steroid hormones, beside their classical genomic mechanism of action, exert rapid, non genomic effects in different cell types. These effects are mediated by still poorly characterized plasma membrane receptors that appear to be distinct from the classic intracellular receptors. In the present study we evaluated the non genomic effects of estradiol (17βE₂) in human sperm and its effects on sperm stimulation by extracellular ATP, a potent activator of sperm acrosome reaction. In human sperm 17βE₂ induced a rapid increase of intracellular calcium (Ca²⁺) concentrations dependent on an influx of Ca²⁺ from the extracellular medium. The monitoring of the plasma membrane potential variations induced by 17βE₂ showed that this steroid induces a rapid plasma membrane hyperpolarization that was dependent on the presence of Ca²⁺ in the extracellular medium since it was absent in Ca²⁺ free-medium. When sperm were pre-incubated in the presence of the K⁺ channel inhibitor tetra-ethylammonium, the 17βE₂ induced plasma membrane hyperpolarization was blunted suggesting the involvement of K⁺ channels in the hyperpolarizing effects of 17βE₂. Extracellular ATP induced a rapid plasma membrane depolarization followed by acrosome reaction. Sperm pre-incubation with 17βE₂ inhibited the effects of extracellular ATP on sperm plasma membrane potential variations and acrosome reaction. The effects of 17βE₂ were specific since its inactive steroisomer 17αE₂ was inactive. Furthermore the effects of 17βE₂ were not inhibited by tamoxifen, an antagonist of the classic 17βE₂ intracellular receptor.     

Kinetics of xylem loading,membrane potential maintenance,and sensitivity of K+‐permeable channels to reactive oxygen species: physiological traits that differentiate salinity tolerance between pea and barley

Salt sensitive (pea) and salt tolerant (barley) species were used to understand the physiological basis of differential salinity tolerance in crops. Pea plants were much more efficient in restoring otherwise depolarized membrane potential thereby effectively decreasing K⁺ efflux through depolarization‐activated outward rectifying potassium channels. At the same time, pea root apex was 10‐fold more sensitive to physiologically relevant H₂O₂ concentration and accumulated larger amounts of H₂O₂ under saline conditions. This resulted in a rapid loss of cell viability in the pea root apex. Barley plants rapidly loaded Na⁺ into the xylem; this increase was only transient, and xylem and leaf Na⁺ concentration remained at a steady level for weeks. On the contrary, pea plants restricted xylem Na⁺ loading during the first few days of treatment but failed to prevent shoot Na⁺ elevation in the long term. It is concluded that superior salinity tolerance of barley plants compared with pea is conferred by at least three different mechanisms: (1) efficient control of xylem Na⁺ loading; (2) efficient control of H₂O₂ accumulation and reduced sensitivity of non‐selective cation channels to H₂O₂ in the root apex; and (3) higher energy saving efficiency, with less ATP spent to maintain membrane potential under saline conditions.     

Do thyroid hormones function in insects?

Earlier work demonstrated that phenoxy-phenyl compounds such as fenoxycarb and thyroxine mimicked the effects of JH III in causing a reduction in volume of the follicle cells of Locusta migratoria. While these compounds were only moderately effective, a derivative of thyroxine, 3,3',5-triiodothyronine (T3) was as effective as JH III, and T3 has been shown to bind to the same membrane receptor and activate the same pathway as JH III. The current paper shows that other thyroxine derivatives vary in activity. 3,3', 5'-Triiodothyronine (reverse T3) is inactive. 3,5-Diiodothyronine (T2) is more active than JH III, while its relatives (iodines at 3', 5' or at 3,3') are inactive. When follicles are exposed in vitro to rhodamine conjugated T3, the fluorescent compound can be seen to enter the cells and accumulate there: this process is inhibited by cycloheximide or by a temperature of 0 degrees C. The accumulation is antagonised by JH III but not JH I (which does not bind to the JH III membrane receptor) and by an antiserum raised against the putative membrane receptor protein. The action of T3, but not T2, is inhibited by 6-n-propyl-2-thiouracil or by aurothioglucose, both known to inhibit deiodinases. The activity of T3, but not of T2, increases with time of exposure to the follicle cells. These facts suggest that T3 enters the cells by receptor mediated endocytosis and is converted to a more active compound. Immunoreactivity to T3, but not thyroxine, can be detected in the haemolymph of locusts, and the titre varies slightly with the gonotrophic cycle. The food shows immunoreactivity for both thyroxine and T3. These findings suggest that thyroid hormones are ingested by locusts and have the potential to be used as hormonal signals in the control of egg production.     

Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8

Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with K_m values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X_1–7 and P2Y_2,4,11 receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y_1,12,13 receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y₆ receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0–8.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.     

Distinct ONE-GC transduction modes and motifs of the odorants: Uroguanylin and CO2

In a subset of the olfactory sensory neurons ONE-GC^$ membrane guanylate cyclase is a central component of two odorant-dependent cyclic GMP signaling pathways. These odorants are uroguanylin and CO₂. The present study was designed to decipher the biochemical and molecular differences between these two odorant signaling mechanisms. The study shows (1) in contrast to uroguanylin, CO₂ transduction mechanism is Ca²⁺-independent. (2) CO₂ transduction site, like that of uroguanylin-neurocalcin δ, resides in the core catalytic domain, aa 880-1028, of ONE-GC. (3) The site, however, does not overlap the signature neurocalcin δ signal transduction domain, ⁹⁰⁸LSEPIE⁹¹³. Finally, (4) this study negates the prevailing concept that CO₂ uniquely signals ONE-GC activity (Sun et al. [19]; Guo et al. [21]). It demonstrates that it also signals the activation of photoreceptor membrane guanylate cyclase ROS-GC1. These results show an additional new transduction mechanism of the membrane guanylate cyclases and broaden our understanding of the molecular mechanisms by which different odorants using a single guanylate cyclase can regulate diverse cyclic GMP signaling pathways.     

Silica Nanohybrid Membranes with High CO2 Affinity for Green Hydrogen Purification

An effective separation of CO₂ from H₂ can be achieved using currently known polyethylene oxide (PEO)‐based membranes at low temperatures but the CO₂ permeability is inadequate for commerical operations. For commercial‐scale CO₂/H₂ separation, CO₂ permeability of these membranes must be significantly enhanced without compromising CO₂/H₂ selectivity. We report here exceptional CO₂/H₂ separation properties of a nanohybrid membrane comprising polyethylene glycol methacrylate (PEGMA) grafts on an organic‐inorganic membrane (OIM) consisting of a low molecular weight polypropylene oxide (PPO)‐PEO‐PPO diamine and 3‐glycidyloxypropyltrimethoxysilane (GOTMS), an alkoxysilane. The CO₂ gas permeability of this nanohybrid membrane can reach 1990 Barrer with a CO₂/H₂ selectivity of 11 at 35 °C for a mixed gas mixture comprising 50% CO₂ ‐ 50% H₂ at 3.5 atm. The transformation of the inorganic silica phase from a well‐dispersed network of finely defined nanoparticles to rough porous clusters appears to be responsible for this OIM membrane exceeding the performance of other state‐of‐the‐art PEO‐based membranes.     

Feasibility of supercritical CO2 treatment for controlling biofouling in the reverse osmosis process

Physical cleaning and/or chemical cleaning have been generally used to control biofouling in the reverse osmosis (RO) process. However, conventional membrane cleaning methods to control biofouling are limited due to the generation of by-products and the potential for damage to the RO membranes. In this study, supercritical carbon dioxide (SC CO₂) treatment, an environmentally friendly technique, was introduced to control biofouling in the RO process. SC CO₂ (100 bar at 35°C) treatment was performed after biofouling was induced on a commercial RO membrane using Pseudomonas aeruginosa PA01 GFP as a model bacterial strain. P. aeruginosa PA01 GFP biofilm cells were reduced on the RO membrane by >8 log within 30 min, and the permeate flux was sufficiently recovered in a laboratory-scale RO membrane system without any significant damage to the RO membrane. These results suggest that SC CO₂ treatment is a promising alternative membrane cleaning technique for biofouling in the RO process.     

Short-term effects of thyroid hormones during development: Focus on signal transduction

Extranuclear or nongenomic effects of thyroid hormones are mediated by receptors located at the plasma membrane or inside cells, and are independent of protein synthesis. Recently the αVβ3 integrin was identified as a cell membrane receptor for thyroid hormones, and a wide variety of nongenomic effects have now been shown to be induced through binding of thyroid hormones to this receptor. However, also other thyroid hormone receptors can produce nongenomic effects, including the cytoplasmic TRα and TRβ receptors and probably also a G protein-coupled membrane receptor, and increasing importance is now given to thyroid hormone metabolites like 3,5-diiodothyronine and reverse T₃ that can mimick some nongenomic effects of T₃ and T₄. Signal transduction from the αVβ3 integrin may proceed through at least three independent pathways (protein kinase C, Src or mitogen-activated kinases) but the details are still unknown. Thyroid hormones induce nongenomic effects on at least three important Na⁺-dependent transport systems, the Na⁺/K⁺-ATPase, the Na⁺/H⁺ exchanger, and amino acid transport System A, leading to a mitogenic response in embryo cells; but modulation of the same transport systems may have different roles in other cells and at different developmental stages. It seems that thyroid hormones in many cases can modulate nongenomically the same targets affected by the nuclear receptors through long-term mechanisms. Recent results on nongenomic effects confirm the old theory that the primary role of thyroid hormones is to keep the steady-state level of functioning of the cell, but more and more mechanisms are discovered by which this goal can be achieved.     

Prostaglandin E2 facilitates subcellular translocation of the EP4 receptor in neuroectodermal NE-4C stem cells

Prostaglandin E2 (PGE₂) is a lipid mediator released from the phospholipid membranes that mediates important physiological functions in the nervous system via activation of four EP receptors (EP1-4). There is growing evidence for the important role of the PGE₂/EP4 signaling in the nervous system. Previous studies in our lab show that the expression of the EP4 receptor is significantly higher during the neurogenesis period in the mouse. We also showed that in mouse neuroblastoma cells, the PGE₂/EP4 receptor signaling pathway plays a role in regulation of intracellular calcium via a phosphoinositide 3-kinase (PI3K)-dependent mechanism. Recent research indicates that the functional importance of the EP4 receptor depends on its subcellular localization. PGE₂-induced EP4 externalization to the plasma membrane of primary sensory neurons has been shown to play a role in the pain pathway. In the present study, we detected a novel PGE₂–dependent subcellular trafficking of the EP4 receptor in neuroectodermal (NE-4C) stem cells and differentiated NE-4C neuronal cells. We show that PGE₂ induces EP4 externalization from the Golgi apparatus to the plasma membrane in NE-4C stem cells. We also show that the EP4 receptors translocate to growth cones of differentiating NE-4C neuronal cells and that a higher level of PGE₂ enhances its growth cone localization. These results demonstrate that the EP4 receptor relocation to the plasma membrane and growth cones in NE-4C cells is PGE₂ dependent. Thus, the functional role of the PGE₂/EP4 pathway in the developing nervous system may depend on the subcellular localization of the EP4 receptor.     

Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach,Diploptera punctata

The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of Dippu-AstR. And we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted in elevated levels of Ca²⁺ and cAMP, which suggests that Dippu-AstR can act through the Gα_q and Gα_s protein pathways. The study on the target of AST action reveals that FGL/AST affects JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway.     

Interaction between ubiquinone and ATPase in mitochondrial membranes

The extraction of ubiquinone from mitochondrial membranes produces alterations of ATPase activity including a reversible loss of oligomycin sensitivity which is restored by long-chain Q-homologs. Short-chain ubiquinones like Q₃ produce a loss of oligomycin and dicyclohexylcarbodiimide (DCCD) sensitivity in submitochondrial particles. The effect shows uncompetitive or noncompetitive kinetics with respect to oligomycin or DCCD respectively. Long-chain ubiquinones have a competitive effect with Q₃, thus restoring oligomycin sensitivity; they behave, however, in about the same way as Q₃ in lowering the DCCD sensitivity in submitochondrial particles. On the basis of these observations we suggest that ubiquinone may be a physiological modulator of ATPase activity in the mitochondrial membrane.Abbreviations used: BHM, beef heart mitochondria; DCCD, dicyclohexylcarbodiimide; ETP, electron transfer particles (submitochondrial particles); Q, ubiquinone.     

Purification and Characterization of a Major Cell Surface Glycoprotein in Zajdela Ascites Hepatoma Cells Which Displays a Potent Concanavalin A Receptor Activity

A major cell surface sialoglycoprotein with Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells. The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation with NaIO₄ followed by reduction with NaB³H₄. Surface-labeled glycoproteins were released by short incubations with TPCK-trypsin at 37°C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP II₂, was then purified by chromatography on DEAE-cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS-polyacrylamide gels, isoelectric focusing, rechromatography on DEAE-cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,000 daltons. The location of GP II₂on the cell surface was confirmed by the fact that it could be labeled metabolically with, D-(³H) glucosamine and externally through the nonpenetrating periodate-NaB³H₄ system. GP II₂could not be removed from the cell surface by high salt concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP II₂could be an integral protein. Analysis of the carbohydrate composition of GP II₂ revealed galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both O- and N-linked to the polypeptide chain, most of them being O-linked. Finally, GP II₂has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.     

前列腺素核受体系统信号转导及基因表达调控

脂肪酸和前列腺素等脂代谢的产物不仅通过膜受体起作用,也可以通过与核受体结合来调节基因表达.前列腺素I₂(PGI₂)既可以与G蛋白偶联的细胞表面IP受体起作用,也可以通过核受体过氧化物酶体增殖因子活化受体（PPARs）发挥生物学功能.前列腺素E₂(PGE₂)的受体（EPs）不仅仅在质膜上有,最近在核膜上也发现了EPs受体.前列腺素核受体介导的信号转导途径与膜受体介导的信号途径不同,对于基因转录的调控机制也不同.     

Lysophosphatidic acid induces inositol phosphate and calcium signals in exocrine cells from the avian nasal salt gland

We tested lysophosphatidic acid (LPA), known to induce inositol phosphate generation and calcium signals as well as rearrangements of the cytoskeleton and mitogenic responses in fibroblasts, for its ability to activate phospholipase C in an exocrine cell system, the salt-secreting cells from the avian nasal salt gland. LPA (>10 nmol/l) caused the generation of inositol phosphates from membrane-bound phosphatidylinositides. The resulting calcium signals resembled those generated upon activation of muscarinic receptors, the physiological stimulus triggering salt secretion in these cells. However, close examination of the LPA-mediated calcium signals revealed that the initial calcium spike induced by high concentrations of LPA (>10 μmol/l) may contain a component that is not dependent upon generation of inositol (1,4,5)-trisphosphate (Ins(1,4,5)P₃) and may result from calcium influx from the extracellular medium induced by LPA in a direct manner. Low concentrations of LPA (<10 μmol/l), however, induce inositol phosphate generation, Ins(1,4,5)P₃-mediated release of calcium from intracellular pools and calcium entry. These effects seem to be mediated by a specific plasma membrane receptor and a G protein transducing the signal to phospholipase C in a pertussis-toxin-insensitive manner. Signaling pathways of the muscarinic receptor and the putative LPA-receptor seem to merge at the G-protein level as indicated by the fact that carbachol and LPA trigger hydrolysis of the same pool of phosphatidylinositol (4,5)-bisphosphate (PIP₂) and mobilize calcium from the same intracellular stores.